Method for preparing a composition by extraction of mother-of-pearl, composition obtained by said method and use thereof in cosmetics and dermatology

ABSTRACT

A method for preparing a composition, comprising a) reducing mother-of-pearl into powder with grain size distribution ranging between about 1 and about 300 μm; b) placing the obtained powder in close contact with an extracting agent in the form of a hydroglycol solution of at least a collagen, of at least a proteoglycan or a mixture thereof; then c) in collecting the extraction mixture, formed by the close contact, constituting the desired composition. The composition obtainable by said method has very interesting properties relative to the skin and/or skin appendages, in particular tissue-regenerating properties, enabling for instance improved wound healing, and anti-ageing properties for preventing and/or visibly reducing effects related to skin and/or skin appendage ageing.

SUMMARY OF THE INVENTION

The present invention relates to a method for preparing a compositioncomprising all the components of mother-of-pearl, at least one collagenand at least one proteoglycan, to the composition which can be obtainedusing this method and to the use of the latter both in thepharmaceutical domain and in the domain of cosmetics, in particular forcombating the effects of aging of the skin and/or of superficial skingrowths.

BACKGROUND OF THE INVENTION

Mammals are protected from the outside environment by a barrierconsisting of the skin, which is a highly structured tissue composed ofseveral layers, but sensitive to attacks due to the variations in theextracorporeal environment. This situation is unique in the animalkingdom since fish and frogs secrete mucus, birds are covered withfeathers, and submammalian and mammalian vertebrates, except currentHominidae, are covered with hair.

Humans have lost these protective attributes: they have a skin whichmust be protected and which can be stimulated in order to help it tocombat attacks and aging.

In particular, it is known that aging is a physiological phenomenonwhich results in particular in thinning of the skin and a loss ofelasticity, leading in particular to the appearance of more or less deepwrinkles. Loosening or drying-out of the surface and anarchicpigmentation may also be observed.

The skin comprises three layers: the epidermis, the dermis and, deepdown, the hypodermis.

The outermost protective envelope of the skin, the epidermis, whichtightly covers the dermis, consists, at the surface, of the stratumcorneum. The stratum corneum is made of two layers: the stratumdisjunctum at the surface, the particularity of which is desquamation,and the stratum compactum, the deepest, which plays the role of abarrier. The epidermis, which is easy to observe macroscopically sinceit is at the surface, has been the subject of many studies. Knownresponses are those caused by certain agents of the extracorporealenvironment or those resulting from the application of active substancesof diverse origins. The cells of the epidermis result from the activityof the cells of the basal layer which lies on a basal membraneseparating the epidermis from the dermis.

The dermis results from the biosynthetic activity of fibroblasts, whichproduce the constituents of the extracellular matrix. The latter is madeup of four major families of macromolecules: collagens, elastin,structural glycoproteins and proteoglycans. The dermis has the abilityto respond to the signals given out by the epidermis, which, inresponse, also sends signals to the epidermis. In general, exchangesexist between these various dermal and epidermal layers of the skin,which are intended to ensure cell renewal and the cohesion andmoisturization of the outer layers.

Many active agents have been proposed for preventing or delaying theeffects of aging.

Among these, mother-of-pearl has been used since the beginning of timein aesthetics and in conventional pharmacopeiae. Moreover,mother-of-pearl is known for its bone-regenerating properties.

It is in particular known that mother-of-pearl, or conchiferousaragonite, is a biogenic mineralized formation; it consists of anorganic matrix of fibrous and nonfibrous substances representingapproximately 1.7 to 2% of the total mass (Taylor et al, Bulletin of theBritish Museum (Natural history) Zoology. Suppl. 3125 pp.+29 Plates,1969) and of calcium carbonate crystallized in orthorhombic form, namedaragonite, combined with trace elements (sodium, magnesium, lanthanum,zinc, bromine, cesium, iron, manganese, chlorine, copper, potassium,calcium, strontium and sulfur). More particularly, the entire organicphase of mother-of-pearl is in the form of an organic matrix composed offibrous proteins, consisting in particular of ancestral collagenslacking hydroxyproline and hydroxylysine, and of nonfibrous proteins.Approximately 50% of the organic matrix of mother-of-pearl iswater-soluble. The remaining 50% can only be obtained afterdecalcification.

However, the products of the prior art, obtained by mixing amother-of-pearl powder with a pulverulent, inert diluent or excipient,as described, for example, in application WO 97/23231, cause undesirablephenomena of skin irritation, due to the presence of the mother-of-pearlin the form of powder (in particular of its major component, aragoniteor CaCO₃). It is therefore imperative that the content ofmother-of-pearl powder in these products should be very limited in orderto avoid these irritation phenomena. These undesirable phenomena ofirritation are in particular caused by the pH, which is too basic, ofthese known products, in contact with the skin. In addition, the knownproducts based on mother-of-pearl powder have specific formulationproblems, in particular stability (breaking of emulsions) and ofadjustment of the pH to a less basic value.

Moreover, application WO 97/24133 describes a method for preparingbiologically active substances from mother-of-pearl, by bringing amother-of-pearl powder into contact with an aqueous solvent chosen frompure, double-distilled or apyrogenic water, optionally supplemented withsalts, from which the water-soluble fraction is then separated so as torecover only the aqueous fraction, therefore essentially lacking thewater-insoluble organic phase and inorganic components ofmother-of-pearl. This method cannot therefore enable, in particular, thepreparation of a composition comprising in particular all of thecomponents of mother-of-pearl, particularly the entire organic phase ofmother-of-pearl.

DETAILED DESCRIPTION OF THE INVENTION

It has now been noted, quite surprisingly and unexpectedly, that theimplementation of a specific method makes it possible to obtain a novelcomposition comprising not only all of the components ofmother-of-pearl, but also an advantageous combination of the latter withcertain compounds which make it possible at least to potentiate theactivity of the components of mother-of-pearl, or even to provide asynergistic effect, for the purpose in particular of preventing and/orvisibly decreasing the effects relating to aging of the skin and/orsuperficial body growths.

A subject of the present invention is thus a method for preparing acomposition, characterized in that it comprises the steps consisting:

-   a) in reducing mother-of-pearl to a powder with a particle size of    between approximately 1 and approximately 300 μm;-   b) in bringing the mother-of-pearl powder thus obtained into close    contact with an extracting agent in the form of an aqueous-glycolic    solution of at least one collagen, or of at least one proteoglycan    or of a mixture thereof; and then-   c) in recovering the extraction mixture formed as a result of the    bringing into close contact, constituting the desired composition.

The present method makes it possible to obtain a composition in the formof the extraction mixture obtained in step b).

The mother-of-pearl used for the implementation of the method accordingto the invention may be obtained from shells of nacreous mollusks and ofsome cephalopods, for example nautilus). In particular, it is obtainedfrom oysters, such as Pinctada maxima.

Raw mother-of-pearl is used, which is free of other polysaccharide- andcalcite-rich shell elements. The starting material is preferably whitemother-of-pearl, otherwise it is necessary to provide a step forremoving the pigments which may cause intolerance.

This is a readily available raw material, the use of which does notimpact negatively on natural populations. In fact, most of these oystersor other nacreous mollusks are farmed.

Furthermore, an additional advantage comes from the fact that the rawmaterial can be obtained from oyster shells which have produced pearls;specifically, a pearl oyster is removed from the productive pool afterhaving produced at most three pearls in succession, although it has athick layer of mother-of-pearl of excellent quality (grade A). Thepresent invention therefore provides an extra opportunity for usingmother-of-pearl downstream of pearl farming.

The particle size of the mother-of-pearl powder used for carrying outthe present method is between approximately 1 and approximately 300 μm,as measured with conventional means within the scope of those skilled inthe art, such as the sieving technique and/or the LASER-readingtechnique.

According to a particular embodiment of the method according to theinvention, the mother-of-pearl is reduced to a powder with a particlesize of between approximately 50 and approximately 100 μm.

According to another particular embodiment, the mother-of-pearl isreduced to a powder with a particle size of between approximately 15 andapproximately 50 μm, which makes it possible to improve the yield byapproaching the size of a crystalline unit (for example, the elementalunit of mother-of-pearl from Pinctada maxima is the biocrystal, a veryvoluminous hexagonal crystal of aragonite of 9 to 12 μm).

The following procedure is in particular carried out in order to reducethe mother-of-pearl to powder according to step a) of the presentmethod.

In a first phase, the outer part of the shell (periostracum) is removedfrom the raw mother-of-pearl by grinding or any other nondenaturingmethod.

The sheets of mother-of-pearl are then reduced to fragments by crushingso as to then be able to perform micronization. The fragments intendedto be micronized are preferably between approximately 2 andapproximately 5 centimeters in length and are approximately 0.3centimeters thick.

In a second phase, the fragments are micronized.

The mother-of-pearl can be used without prior decontamination. In thecase of prior decontamination, a very rapid decontaminating wash isperformed, without soaking the fragments, in a solution of sodiumhypochlorite at 6.6% of active chlorine (12°). Drying must be carriedout extemporaneously so as to remove all traces of water.

The grinding must be done dry in zirconium jars reserved exclusively forthis purpose, washed (water containing bleach, rinse, then wash withdistilled water) and hot-sterilized beforehand. The crushing is carriedout with zirconium balls, themselves sterilized.

The raw mother-of-pearl reduced to powder can be sterilized in twodifferent ways:

-   -   sterilization by irradiation with γ-rays, 2.5 Mrad;    -   hot-sterilization for 1 to 2 hours in an autoclave at 100° C.        The mother-of-pearl is degraded (inorganic matrix and organic        matrix) only from approximately 250° C. (Balmain, Hannoyer and        Lopez, “Fourier transform infrared spectroscopy (FTIR) and X-ray        diffraction analyses of mineral and organic matrix during        heating of Mother of Pearl (nacre) from the shell of the mollusc        Pinctada Maxima”, J. Biomed. Mater. Res. (Appl. Biomater.), vol.        48(5): 749-754, 1999). This sterilization does not therefore        destroy its various components.

Step b) of the method according to the invention consists in bringingthe mother-of-pearl powder described above into close contact with anextracting agent in the form of an aqueous-glycolic solution of at leastone collagen, of at least one proteoglycan or of a mixture thereof.

According to the invention, the term “aqueous-glycolic solution” isintended to mean a solution of the collagen and the proteoglycanobtained using an aqueous-glycolic solvent, i.e., in general, a solventin the form of a mixture of water and at least one glycol.

The term “glycol” is intended to mean, in a known manner, any compoundhaving two alcohol functions. In particular, the glycol which may beused is chosen from the group consisting of ethylene glycol, propyleneglycol, butylene glycol and mixtures thereof.

The water which may be used to form the aqueous-glycolic solvent may bepure, double-distilled, apyrogenic or demineralized water, or elsedeionized water.

The water:glycol weight ratio in the aqueous-glycolic solvent ispreferably between approximately 1:100 and approximately 100:1, and moreparticularly between approximately 1:1 and approximately 20:1.

The extracting agent is preferably an aqueous-glycolic solution of atleast one collagen.

The collagen which may be used in aqueous-glycolic solution in theextracting agent according to the invention may be any collagenconstituting the intracellular substance of the connective tissue,available in the animal kingdom, known to those skilled in the art.

Most particularly, use is preferably made of a marine collagen, i.e. acollagen derived from an organism of marine origin, such as marinevertebrates. In particular, marine collagen is the main constituent ofthe connective tissues of fish, in which it fulfills an essential rolein the structure of the skin, muscles, tendons and ligaments.

Mention may be made more particularly of the marine collagen“PANCOGENE^(R) MARIN”, as sold by the company Gattefossé (Saint Priest,France), the INCI name of which is “Soluble Collagen” (referencing inJapan: “water soluble collagen”; MHV: 20800CZY00100000). It is acollagen extracted from skins of unprotected species of fish from warmseas, belonging to the class Teleostei.

Mention may also be made of the marine collagen “COLLAGEN NATIFMARIN—Code 690”, as marketed by the company Laboratoire Industriel deBiologie (Soisy Sous Montmorency, France), the CTFA name of which is“Amino Collagen Amino Acid”. It is a water-soluble acid collagenextracted from fish skins.

Preferably, a collagen concentration is used which is betweenapproximately 0.0001 and approximately 50% by weight, and moreparticularly between approximately 0.01 and approximately 15% by weight,relative to the total weight of the extracting agent.

The extracting agent is also preferably an aqueous-glycolic solution ofat least one proteoglycan.

The proteoglycan which may be used in aqueous-glycolic solution in theextracting agent according to the invention may be any proteoglycanknown to those skilled in the art, in particular any proteoglycan notcontaining sulfur.

In particular, the proteoglycan is chosen from the group consisting ofhyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate,keratan sulfate and mixtures thereof.

Hyaluronic acid is most particularly preferred, such as that marketed bythe company Laboratoire Industriel de Biologie (Soisy Sous Montmorency,France).

The proteoglycan concentration used is preferably between approximately0.0001 and approximately 40% by weight, and more particularly betweenapproximately 0.01 and approximately 10% by weight, relative to thetotal weight of the extracting agent.

Of course, the extracting agent may also comprise any additionalcompound known to those skilled in the art, which is suitable f or theextraction step itself or in view of the possible uses of thecomposition prepared by the present method, such as those describedhereafter. The extracting agent may thus, for example, also comprisecomplexing agents, such as EDTA (ethylenediaminetetraacetic acid).

According to a particularly preferred embodiment of the presentinvention, the mother-of-pearl powder is brought into close contact withthe extracting agent according to step b) by preparing a mixture,consisting of the mother-of-pearl powder and the extracting agent, suchthat it comprises, relative to its total weight, approximately 20 toapproximately 60% by weight of mother-of-pearl powder obtained in stepa), as described above, and the remainder as extracting agent asdescribed above.

The mother-of-pearl powder can be brought into close contact with theextracting agent, according to step b) of the present method, inparticular by suspending the mother-of-pearl powder in the extractingagent, with vigorous and homogeneous mechanical stirring, so as to allowan extraction coating of the mother-of-pearl particles.

The procedure may, for example, be carried out at ambient temperature,i.e. at a temperature of about 20° C. for approximately 1 hour, but thedurations and temperatures may be adjusted, by those skilled in the art,in particular depending on the starting particle size of themother-of-pearl powder.

According to another embodiment, step b) for bringing about contact maybe carried out by passing the extracting agent, under pressure, throughthe mother-of-pearl powder, which has been immobilized. Thisimmobilization may, for example, be effected using a column, of the HPLCtype, optionally in a mixture with fillers which allow better diffusionof the extracting agent and avoid compaction of the mother-of-pearlpowder.

Without wishing, however, to be linked to any particular theory, it isthought that the presence of the collagen and of the proteoglycan in theextracting agent, in particular of the marine collagen and of thehyaluronic acid, would promote extraction of related compounds presentin the mother-of-pearl, via a physicochemical phenomenon of affinity,with, in addition, potentiation of the effects of the components thusextracted from the mother-of-pearl. In particular, it is thought thatextraction of the ancestral collagens from the mother-of-pearl would bepromoted by the presence of the marine collagens and extraction of boththe adhesion proteins (decorin and cytokines) and the proteoglycans fromthe mother-of-pearl would be promoted by the presence of theproteoglycan chosen.

Be that as it may, the close contact is preferably brought about, for agiven temperature, for a period of time sufficient to produce avirtually complete extraction. According to the invention, the term“virtually complete extraction” is intended to mean the extraction ofall the extractable components from the mother-of-pearl when it isbrought into contact with the extracting agent.

In other words, the production of an equilibrium for the totalconcentrations in particular of collagens or of proteoglycans in theaqueous-glycolic liquid phase corresponds to achieving said “virtuallycomplete extraction”.

It is therefore within the scope of those skilled in the art todetermine the “virtually complete” nature of such extraction, forexample by regularly measuring these total collagen or proteoglycanconcentrations in the aqueous-glycolic liquid phase.

Be that as it may, the step for bringing about contact may comprise,before it ends, a period during which the suspension of themother-of-pearl powder in the extracting agent is left to stand(stirring arrested).

At the end of step b) for bringing about contact, the extractionmixture, formed as a result of the bringing into close contact, whichconstitutes the desired composition, is recovered.

This composition, in the form of an aqueous-glycolic suspension, may bestored as it is before use. For practical reasons of subsequentimplementation (storage, transport, formulation, etc.) it is alsopossible to separate the liquid phase of this composition from the solidphase.

Thus, according to one variant, the method according to the invention ischaracterized in that, at the end of step b), the extraction mixture,formed as a result of the bringing into close contact, which constitutesthe desired composition, is recovered and the liquid phase of thecomposition is separated from the solid phase by means known to thoseskilled in the art, such as ultra-filtration means, tangentialfiltration means, etc.

It is understood that the solid and liquid phases thus separated,together, constitute the composition prepared as described above. It maythus be envisioned that, in a subsequent formulation using thiscomposition, with pharmaceutically or cosmetically suitable excipients,the composition may be reconstituted at any step of the formulation, byaddition of the respective solid and liquid phases, simultaneously orseparated over time, for reasons of convenience of formulation. Giventhe above, it is understood that a subject of the present invention isalso the liquid phase as obtained at the end of the separation stepdescribed above. Similarly, a subject of the present invention is alsothe solid phase as obtained at the end of the separation step describedabove.

A subject of the present invention is also a novel composition which canbe obtained using the method as described above (comprising the solidand liquid phases, with or without a separation step as describedabove).

In particular, this composition is characterized in that it comprises atleast, in the form of an aqueous-glycolic suspension of a liquid phaseand of a solid phase:

-   -   aragonite (CaCO₃);    -   trace elements chosen from the group consisting of sodium,        magnesium, lanthanum, zinc, bromine, cesium, iron, manganese,        chlorine, copper, potassium, calcium, strontium, sulfur and        mixtures thereof;    -   fibrous proteins from mother-of-pearl, in particular ancestral        collagens from mother-of-pearl, related to the marine collagens        of the extracting agent (see FIG. 1 described below);    -   nonfibrous proteins from mother-of-pearl, in particular proteins        related to adhesion proteins, such as decorin (see FIG. 2        described below);    -   at least one collagen not derived from mother-of-pearl and/or at        least one proteoglycan not derived from mother-of-pearl.

It is, of course, understood that, according to the invention, theexpressions “collagen not derived from mother-of-pearl” and“proteoglycan not derived from mother-of-pearl” are intended to mean,respectively, the collagen and the proteoglycan used for the extractingagent of step b) of the present method. Similarly, the expression“aqueous-glycolic suspension” is explained by the fact that thecomposition according to the invention comprises not only solublecomponents (in aqueous-glycolic solution) but also insoluble components(in particular aragonite).

Moreover, the preferences stated above for the method according to theinvention apply, of course, to this composition.

Thus, in particular, the collagen not derived from mother-of-pearl inthe composition according to the invention is preferably a marinecollagen, and more particularly a marine collagen chosen from the groupconsisting of “PANCOGENE^(R) MARIN”, “COLLAGENE NATIF MARIN—Code 690”and mixtures thereof. Similarly, the proteoglycan not derived frommother-of-pearl may be any proteoglycan known to those skilled in theart, in particular any proteoglycan not containing sulfur, and is inparticular chosen from the group consisting of hyaluronic acid,chondroitin sulfate, dermatan sulfate, eparan sulfate, keratan sulfateand mixtures thereof, and is more particularly hyaluronic acid.

As shown in greater detail in the examples below, this composition hasproperties which are very advantageous with respect to the skin and/orsuperficial body growths, in particular tissue-regenerating properties,allowing for example improved wound healing, and anti-aging propertiesmaking it possible to prevent and/or visibly decrease the effectsrelated to aging of the skin and/or superficial body growths. Its actionon the various cell types of the skin is one of repair and of regulationof the physiological balance between its various constituents. Moreparticularly, its organic and inorganic components act in particular, atseveral levels, on keratinocyte metabolism. This composition allowsrestructuring of the epidermis, contributing to better protection of thedeepest layers. The epidermis becomes more resistant and deeper andinteracts continually with the dermis. In addition, this compositionallows enrichment in elastin and collagens. The skin is more elastic andfirmer. It is resistant and its components are renewed at a sustainedrhythm. In addition, this composition stabilizes synthesis of pigmentsand promotes microcirculation. Finally, this composition has theadvantage of being totally innocuous and that of having ananti-inflammatory, and therefore soothing, effect.

Thus, a subject of the present invention is also a pharmaceuticalcomposition, characterized in that it comprises, as active principle,the novel composition as described above, which can be obtained usingthe method according to the invention, and a pharmaceutically acceptableexcipient.

The pharmaceutically acceptable excipient is preferably an excipientsuitable for dermatological application.

A subject of the present invention is also the use of this novelcomposition as described above, which can be obtained using the methodas described above, for producing a medicinal product intended for thetreatment of tissue regeneration disorders of the skin and/orsuperficial body growths.

Moreover, a subject of the present invention is the use of the novelcomposition as described above, which can be obtained using the methodas described above, for producing a medicinal product intended for thetreat-ment of disorders of the skin and/or superficial body growthsrelated to aging.

Finally, a subject of the present invention is the use of the novelcomposition as described above, which can be obtained using the methodas described above, for producing a medicinal product intended for thetreat-ment of inflammatory skin manifestations.

A subject of the present invention is also a cosmetic composition,characterized in that it comprises, as cosmetically active principle,the novel composition as described above, which can be obtained usingthe method according to the invention, and a cosmetically acceptableexcipient.

Any cosmetic excipient known to those skilled in the art may be used.

Of course, the cosmetic composition according to the invention may alsocontain ingredients of the cosmetic type, known to those skilled in theart, such as moisturizers, softeners, or chemical or organic screeningagents.

The pharmaceutical and cosmetic compositions described above may, inparticular, be in the form of a cream, an ointment, a gel, a lotion, anoil-in-water emulsion or a water-in-oil emulsion, or may be combinedwith any pharmaceutically or cosmetically acceptable vector, such asliposomes.

In addition, a subject of the present invention is the use of the novelcomposition as described above, which can be obtained using the methodaccording to the invention, for producing a cosmetic compositionintended for cosmetic treatment for tissue regeneration of the skinand/or superficial body growths.

Moreover, a subject of the present invention is the use of the novelcomposition as described above, which can be obtained using the methodaccording to the invention, for producing a cosmetic compositionintended for the cosmetic treatment of modifications related to aging ofthe skin and/or superficial body growths.

Finally, a subject of the present invention is a method of cosmetictreatment for tissue regeneration of the skin and/or superficial bodygrowths, characterized in that the novel composition as described above,which can be obtained using the method according to the invention, isapplied to the skin and/or superficial body growths.

Furthermore, a subject of the present invention is a method of cosmetictreatment of modifications related to aging of the skin and/orsuperficial body growths, characterized in that the novel composition asdescribed above, which can be obtained using the method according to theinvention, is applied to the skin and/or superficial body growths.

FIGS. 1 and 2 represent a Western blot of a composition prepared usingthe method according to the invention.

FIG. 3 represents a histogram of the overall amino acid composition ofthe protein phase of the composition prepared using the method accordingto the invention.

FIGS. 4 and 5 are photographs illustrating the effect of the compositionprepared according to the invention on cytokeratin synthesis by humankeratinocytes.

FIGS. 6 and 7 are photographs illustrating the effect of the compositionprepared according to the invention on human keratinocytes in culturesubjected to estradiol withdrawal.

FIGS. 8 and 9 are photographs illustrating the effect of the compositionprepared according to the invention con cytokeratin synthesis by humankeratinocytes in culture subjected to estradiol withdrawal.

FIGS. 10 and 11 are photographs illustrating the effect of thecomposition prepared according to the invention on elastin synthesis byhuman fibroblasts.

FIGS. 12 and 13 are photographs illustrating the effect of estrogens onhuman fibroblasts.

FIGS. 14 and 15 are photographs illustrating the effect of thecomposition prepared according to the invention on human fibroblastssubjected to estradiol withdrawal.

FIG. 16 is a histogram illustrating the effect of the compositionprepared according to the invention on melanocytes, through themodification of the amount of melanin taken up by keratinocytes culturedin the presence of the composition.

FIGS. 17 and 18 are photographs illustrating the effect of thecomposition prepared according to the invention on the amount of melanintaken up by cultured human keratinocytes.

FIG. 19 is a histogram illustrating the effect of the compositionprepared according to the invention on interleukin 1 (IL-1) secretion byHL 60 promyelocytic cells.

Finally, FIG. 20 is a histogram illustrating the study of thenoncytotoxicity of the composition prepared according to the invention.

The following examples are intended to illustrate the present inventionbut should in no way be interpreted as possibly restricting the scopethereof.

EXAMPLE 1 Method for Preparing a Composition According to the Invention

The material used should be a material of the type under vacuum,completely airtight, and preferably made of stainless steel. It shouldbe washed and rinsed cleanly beforehand so as to avoid any risk ofbacterial contamination.

1.1) Mother-of-pearl from Pinctada maxima, from which the outer part ofthe shell (periostracum) has already been removed, and which has alreadybeen made into irregular fragments, is used (supplied by the companyPharma Futura 10, rue de Charonne, 75012 PARIS). The size of thefragments is between 2 and 5 centimeters in length and 0.3 centimetersin thickness.

The fragments are given a very rapid decontaminating wash, withoutsoaking, in a solution of sodium hypochlorite at 6.6% of active chlorine(12°) and are then dried extemporaneously in order to remove any traceof water.

The grinding for the micronization must be carried out dry in zirconiumjars reserved exclusively for this purpose, washed (water containingbleach+rinse+distilled water wash) and hot-sterilized beforehand. Thegrinding is carried out using zirconium balls, which are themselvessterilized.

The micronized mother-of-pearl powder thus obtained has a particle sizeof between 50 and 150 μm. It is then sterilized by irradiation withγ-rays, 2.5 Mrad.

The micronized mother-of-pearl powder thus prepared has the followingcharacteristics:

appearance pulverulent color white odor characteristic ofmother-of-pearl arsenic < 1 ppm heavy metals < 10 ppm.Bacteriological Analysis:

number of aerobic microorganisms 1550/g degree of hygrometry 0.3%

1.2) The following mixture is prepared: 400 grams of Pinctada Maximamother-of-pearl powder micronized as described above (particle sizebetween 50 and 150 μm) are gradually added to 600 ml of an extractingagent in the form of an aqueous-glycolic solution (water and ethyleneglycol, respectively, in a 10:1 weight ratio) comprising 0.18% by weightof the marine collagen “PANCOGENE^(R) MARIN” (marketed by the companyGattefossé; Saint Priest, France; INCI name “Soluble Collagen”;referencing in Japan: “water soluble collagen”; MHV: 20800CZY0010000)and 0.2% by weight of hyaluronic acid (marketed by the companyLaboratoire Industriel de Bioiogie; Soisy Sous Montmorency, France),relative to the total weight of the extracting agent, with stirring atapproximately 4 000 rpm and at a temperature of approximately 20° C.Once this has been added, the stirring and temperature are maintainedfor 60 minutes. The stirring must be vigorous and homogeneous in orderto bring all the particles of the mixture into contact and allow anextraction coating.

The mixture is then left to stand for 6 hours.

The composition thus obtained is composed of an off-white particulatesolid phase, in suspension in a liquid phase and at a pH ofapproximately 8.

This composition comprises:

-   -   aragonite (CaCO₃);    -   marine trace elements: sodium, magnesium, lanthanum, zinc,        bromine, cesium, iron, manganese, chlorine, copper, potassium,        calcium, strontium and sulfur (see table 1 below);    -   fibrous proteins: various types of protein related to marine        collagens, which may be considered to be ancestral collagens        from the mother-of-pearl, and the marine collagen “PANCOGENE^(R)        MARIN”;    -   nonfibrous proteins: proteoglycans, including the hyaluronic        acid from the mother-of-pearl and the extracting agent, and also        adhesion proteins from the mother-of-pearl (decorin rp and        cytokine rp; “rp meaning “related peptides or proteins” i.e.        peptides or proteins related, respectively, to decorin and to        cytokine insofar as they are recognized by the antibodies        against the latter molecules).

The technique used to identify the peptides and proteins in the contextof the present invention are those described in the following articles:

-   -   electrophoresis—SDS-Page method: Laenli method, Nature 1970, 227        680-682;    -   immunotransfer: Western blot according to Towbin H. et al.,        1979, Proc. Natl. Acad. Sci. USA 76(9): 4350-4354 and        Burnette W. N. 1981, Analytical Biochemistry 112: 195-203.

The corresponding Western blots are represented in FIGS. 1 and 2.

The amino acid analysis of the protein phase indicates the presence ofaspartic acid, threonine, serine, glutamic acid, glycine, alanine,proline, valine, methionine, isoleucine, leucine, tyrosine,phenylalanine, histidine, lysine and arginine. FIG. 3 represents ahistogram of the overall amino acid composition.

The composition obtained as described above will be named hereafter“composition A”.

TABLE 1 quantitative analysis of the trace elements contained incomposition A Mean concentration Mean concentration Element (μg/g)Element (g/100 g) Sulfur 0.02 Calcium 38.8 Magnesium 2 Lanthanum 0.28Zinc 0.4 Bromine 1.78 Cesium 5.5 Iron 13.6 Manganese 50 Chlorine 296Copper 143 Potassium 582 Strontium 1000 Sodium 5420

EXAMPLE 2 Study of the Activity of Composition A

Histological and cytological scientific research, which uses either invivo animal models or human cell cultures by exploiting microscopy andthe most modern techniques for detecting substances by immunology andimmunocytochemistry, currently allows studies to be carried out on acellular scale, and such a study was performed in order to investigatethe active substances of this composition A.

This study essentially concerned three types of cell insofar as they areknown to play a major role in the structure, functioning andpigmentation of skin tissue: keratinocytes, fibroblasts and melanocytes.Both the keratinocyte and fibroblast cell types have receptors specificfor the active substances of composition A, and in particular of all ofthe components of the mother-of-pearl which are present therein, leadingto a cascade of messages which may reach the level of the deepest layersof the dermis.

The study related not only to their morphological appearance but also tothe behavior of their main components, nuclei and cytoplasm, reflectingtheir synthetic activity and their capacity for renewal. The presence ofcertain active substance and that of secreted structural proteins wasalso identified.

1.1) Activity of Composition A on Keratinocytes: Cytokeratin Synthesisby Human Keratinocytes Cultured in the Presence of Composition A

1.1.1) Method

Keratinocytes are the outermost cells of our body. They constitute ourfirst protective barrier. Keratinocytes intervene by acting passivelyand actively. They produce the passive protection by constituting abarrier which plays the role of a shield. This shield is made from cellswhich are dried-out and anucleated. The strong cohesion of the cellsmakes it possible to provide a very homogeneous system. Keratinocytessynthesize substances, cytokeratins, which they maintain in theircytoplasm in order to give this barrier structure. The cytokeratinsconstitute the internal skeleton of keratinocytes. This internalskeleton gives these cells their volume and increases their capacity forintercellular communication and facilitates melanin uptake. The richerthe epidermis is in cytokeratins, the more effective it is and the moreit gives the skin its young appearance. By facilitating contact betweenthe cells, the cytokeratins prevent loss of the NMS (NaturalMoisturizing Factors, such as ceramides, cholesterol or fatty acids).

The active protection of the skin is related to the fact thatkeratinocytes are the first cells of our body to be in contact with theoutside. They have therefore developed a panoply of components intendedto inform our body of possible changes in the conditions of ourenvironment. To do this, these cells are capable of synthesizing growthfactors intended to stimulate the cells subjacent to the dermis(fibroblasts), in particular PTH related peptide (PTHrp), which issynthesized mainly by keratinocytes and known to be a cellulardifferentiation factor.

The method used was as follows. It includes a rapid semiquantitativetechnique which makes it possible to assess the level of each of thesecreted proteins present in the cell cytoplasm.

4×10⁴ cells are seeded per 0.9 cm² chamber (8-chamber slides, NUNC) andcultured overnight. After rinsing with HBSS buffer, the mediumcontaining 1% of composition A or the control medium (containing 0% ofcomposition A) is added (450 μl per chamber).

The cultures are incubated for 48 h. After the supernatants have beenremoved and the Cultures rinsed, the cells are fixed withparaformaldehyde for 30 min at 40° and then rinsed with PBS buffer.

200 μl of anti-cytokeratin primary antibodies are added. The incubationlasts 30 minutes at room temperature. The supernatants are then removedand the cells are rinsed with PBS.

200 μl of secondary antibodies which recognize the primary antibody andare coupled to a fluorescent marker, in this instance fluorescein, areadded. After incubation for 30 minutes at room temperature, thesupernatants are removed and the cells are rinsed with PBS. The slidesare then mounted, followed by examination under an inverted fluorescencemicroscope (detection of intracytoplasmic cytokeratins byimmunofluorescence).

The amount of proteins, in this instance of cytokeratins, synthesized bythe cells and thus labeled is proportional to the strength of thefluorescence resulting from the immunolabeling.

1.1.2) Results

As shown in FIGS. 4 and 5, the addition of 0.5% of composition A to thekeratinocyte culture medium has the effect of increasing cytokeratinsynthesis. Composition A restores the activity of keratinocytes from oldskin and also the secretion of cytokeratin by these keratinocytes(action on keratinocytes subject to estradiol withdrawal; see FIGS. 6, 7and 8, 9).

In addition, calcium-dependent PTHrp synthesis by the keratinocytes isstimulated in the presence of composition A. The PTHrp, synthesizedmainly by the keratinocytes, is particularly active in the presence ofthe calcium ion provided by composition A and acts synergistically withthe cytokines of the complete organic matrix of the mother-of-pearlpresent in composition A.

Decorin, a major adhesion protein in skin regeneration, is alsostimulated in the presence of composition A.

Cadherins, calcium-dependent adhesion proteins, are also largelyresponsible for cohesion. They are stimulated in the presence ofcomposition A.

1.2) Activity of Composition a on Fibroblasts, Elastin Synthesis

1.2.1) Elastin Synthesis by Human Fibroblasts Cultured in the Presenceof Composition A

The method used is the same as that described in paragraph 1.1.1) abovefor the study on keratinocytes.

As emerges from FIGS. 10 and 11, composition A at a concentration of 1%strongly stimulates elastin synthesis by human fibroblasts.

1.2.2) Comparison of the Effect of Estrogens and of Biocrystal maxima m.on Human Fibroblasts in Culture (FIGS. 12, 13, 14 and 15).

Relatively inactive mature fibroblasts are very elongated and flat inshape and have dense nuclei. Active fibroblasts have nuclei which arelarge in size and rounded, with voluminous nucleoli, reflecting theactive process of synthesis. The “active fibroblast” type is observed inthe cultures of skin explants taken from a 50-year-old woman undergoinghormone replacement therapy.

Estrogen deprivation in the culture medium causes drastic inactivationof the cells. They are no longer adherent nor confluent, and someexhibit the beginning of pyknosis.

FIGS. 12 and 14 illustrate the effect of composition A on humanfibroblasts from explants from 50-year-old menopausal (therefore withdecreased estrogen secretion) women undergoing replacement therapy.

FIGS. 13 and 15 illustrate the effect of composition A on humanfibroblasts subjected to estradiol withdrawal, from explants from50-year-old menopausal women undergoing replacement therapy.

As emerges from these FIGS. 12 to 15, composition A, added to theculture medium at 1%, completely restores the activity of thefibroblasts having been subjected to the withdrawal. The action ofcomposition A is notable: fibroblasts have a well-developed, very clearnucleus with a prominent nucleolus. Their fusiform cell bodies line upin the same direction and are confluent, allowing cellular exchanges.Similar results were obtained when working on cells from explants ofyoung skin.

1.3) Activity of Composition A on Melanocytes: Modification of theAmount of Melamin Taken Up by Keratinocytes Cultured in the Presence ofComposition A

1.3.1) Method

Melanocytes are the pigmentary cells responsible for melanin synthesis,melanin being the pigment which causes skin color. Melanocytic cellsrepresent approximately 2 to 4% of the total epidermal population. Thehomogeneous color of the skin is due to distribution of the pigment overthe entire surface of the skin by virtue of melanin transfer, in theform of melanosomes, from the secretory cells (melanocytes) to theneighboring keratinocytes which take it up.

The method consists in preparing a keratinocyte/melanocyte coculture andassaying melanin.

Keratinocytes and melanocytes are both cell populations located in theepidermis. They are obtained from a skin sample after removal of thedermis and enzymatic digestion of the epidermis. The epidermal cellsthus isolated are counted in a Coulter Counter (Coultronics) and arethen cultured in a conventional culture medium of the medical Dulbecotype (eagle medium) supplemented with glutamine, streptomycin/penicillinand 10 of fetal calf serum. The percentages are adjusted as a functionof the results obtained. This medium allows survival of melanocytes andkeratinocytes.

After incubation for 4 days in the presence of composition A, the cellsare digested with an NaOH/DMSO mixture and the supernatant is thenrecovered after centrifugation. Reading is performed at 470 nm.

1.3.2) Results

The results are given in table 2 below and represented by the histogramin FIG. 16.

TABLE 2 μg of melanin taken up by keratinocytes Standard Sample 1 Sample2 Sample 3 Mean deviation Control 0.150 0.149 0.148 0.149 0.001Composition A 0.150 0.132 0.140 0.141 0.009 (0.5%) Composition A 0.1390.137 0.143 0.140 0.003 (1%)

The presence of 1 of composition A causes a significant change indistribution of the melanin taken up by the keratinocytes, avoidinglocalized concentrations (see FIGS. 17 and 18).

1.4) Immunocytochemical Demonstration of the Stimulation ofKeratinocytes and of Fibroblasts by Composition A

1.4.1) Method

This is a rapid semiquantitative technique which makes it possible toassess the level of each of the proteins (cytokeratin and elastin)present in the cell cytoplasm. 4×10⁴ cells are seeded per 0.9 cm²chamber (8-chamber slide, NUNC) and cultured overnight. After rinsingwith HBSS buffer, the medium containing 1% of composition A or thecontrol medium (containing 0% of composition A) is added (450 μl perchamber). The cultures are incubated for 48 hours. After thesupernatants have been removed and the cultures rinsed, the cells arefixed with paraformaldehyde for 30 minutes at 4° C. and then rinsed withPBS buffer. 200 μl of anti-cytokeratin and/or anti-elastin antibodiesare added. The incubation lasts 30 minutes at room temperature. Thesupernatants are then removed and the cells are rinsed with PBS. 200 μlof the second antibody conjugated to fluorescein are added. Afterincubation for 30 minutes, the supernatants are removed and the cellsare rinsed with PBS. The slides are then mounted, followed byexamination under an inverted fluorescence microscope. The amounts ofelastin or of cytokeratin synthesized by the cells are proportional tothe strength of the fluorescence.

1.4.2) Results

The addition of 1% of composition A to the keratinocyte culture mediumhas the effect of increasing cytokeratin synthesis by the keratinocytecells of the epidermis. This stimulation is observed considerably from0.5% of composition A.

When the fibroblasts are incubated in vitro in the presence ofcomposition A (at 1%) a very noticeable increase in the strength of thefluorescence is observed, after 48 hours, thus reflecting stimulation ofelastin synthesis by the fibroblasts of the dermis.

1.5) Anti-Inflammatory Activity of Composition A: Assaying ofInterleukin 1 (Il-1) Secreted by HL 60 Promyelocytic Cells Treated withComposition A

During an inflammatory process, migration of a large number ofinflammatory cells from the peripheral blood to the sizes ofinflammation is first observed, followed by considerable secretion atthese sites of certain cytokines and in particular of IL-1. The latteris considered to have a major role.

In this study, IL-1 secretion is measured using a cell line, HL-60, inthe presence of composition A.

The production of IL-1 is amplified by adding phytohemaglutinin (PHA),which is a powerful stimulant of IL-1 secretion, to the culture medium.The IL-1 is quantified via the conventional technique of immunoassay byELISA using a specific antibody directed against human IL-1.

1.5.2) Results

The results are given in table 3 below and represented by the histogramin FIG. 19.

TABLE 3 demonstration of the effect of composition A on the productionof interleukin 1 (IL-1, in picograms) secreted by the promyelocyticcells Standard Sample 1 Sample 2 Sample 3 Mean deviation Control 0.7030.685 0.654 0.681 0.025 Composition A 0.689 0.664 0.570 0.641 0.063 (1%)Composition A 0.494 0.615 0.527 0.545 0.063 (0.5%)

In the presence of 1% of composition A, a significant decrease in IL-1synthesis is therefore observed, thus reflecting a very notableanti-inflammatory effect of composition A. This in vitro effect was alsoobserved when implantation was performed in rats in vivo.

1.6) Toxicity Test. Demonstration of the Noncytotoxicity of CompositionA: MTT Test of Mitochondrial Activity

1.6.1) Method

This a colorimetric test based on reduction of a tetrazolium salt (MTT),by the mitochondrial NADPH reductase of live cells, to a blue-purpleformazan product.

2×10⁴ cells, in 200 μl of culture medium, are seeded into each well(96-well plate, NUNC), and then incubated overnight in order to allowthem to adhere properly to the plastic. After rinsing the cells withserum-free medium, the various concentrations contained in the culturemedium (1%, 0.5% and 0.1% of composition A) or the control medium (0% ofcomposition A), are added at 200 μl/well.

After incubation for 48 hours, the supernatants are removed and thecells are rinsed with HBSS buffer. 100 μl of culture medium containing20% of a solution of MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide;“Thiazolyl Blue”) are added to each well. The reaction takes place for 2hours in an incubator at 37° C. at 5% CO₂.

100 μl of 0.04 N HCl in isopropanol are added to allow bettersolubilization of the formazan crystals, and then the colored reactionis read at a wavelength of 570 nm using a microplate reader. The valuesare also expressed as optical density.

1.6.2) Results

The results are given in table 4 and represented by the histogram inFIG. 20.

TABLE 4 optical density measured Sample Sample Sample Sample SampleSample Standard 1 2 3 4 5 6 Mean deviation Control 0.455 0.466 0.4490.449 0.47 0.472 0.460 0.01 Composition A 0.732 0.796 0.771 0.785 0.8080.768 0.777 0.027 (1%) Composition A 0.669 0.675 0.709 0.7 0.598 0.6940.691 0.016 (0.5%) Composition A 0.643 0.648 0.617 0.624 0.621 0.6280.630 0.013 (0.1%)

The presence of composition A in the culture medium therefore leads to aconsiderable increase in the cellular mitochondrial activity. Thisstimulation of the enzymatic activity is dose-dependent: the higher theconcentration of composition A, the higher the activity. However,whatever the content of composition A in the medium, the mitochondrialactivity of the cells is greatly superior to that of the control cellsnot treated with composition A.

2.7) Conclusions Regarding the Activity of Composition A Evaluated InVitro

Composition A acts directly on the most superficial layers of theepidermis.

The organic and inorganic components of the extract express themselvesat several levels in keratinocyte metabolism by acting on the activityof the cells of the basal layer.

First of all, cell growth, division and differentiation are stimulatedand then specific syntheses by the keratinocytes are amplified, inparticular syntheses of cytokeratins and the mediators intended forcorrect functioning of the dermis, such as for example PTHrp, promotingthe intercellular communication cascade.

The differentiation sequence for basal keratinocytes which arestar-shaped and then granular, is stimulated and controlled without anyincrease in the number of cells.

The controlled increase in the number of keratinocytes improves thestructure of the epidermis and the dermal-epidermal junctions. Therestructuring of the epidermis contributes to better protection of thedeepest layers.

The improvement in specific syntheses, such as those of thecytokeratins, stabilizes pigment syntheses and ensures a physiologicalrepartition of the melanin within the keratinocytes, resulting in thelack of localized concentrations. The improvement in the protection ofthe skin occurs without inducing any abnormal thickening of the skin,while at the same time deepening the sheets of cells layered on thedermal-epidermal junction.

Substances which contribute to improving the response of cells to attackand to inflammation, and also the communication factors which allowphysiological exchanges between the epidermis and dermis, are foundamong the set of mediators secreted by the keratinocytes. The cellularrenewal of the epidermis, which is 4 to 6 weeks, is maintained at itsphysiological rhythm.

Amplification of mediator synthesis increases the recruitment of anentire set of cells, and the decorin provided by composition A at theepidermal level is included among these mediators.

2.7.1) At the Epidermal Level

By activating PTHrp synthesis, composition A acts on the growth anddifferentiation of keratinocytes. This effect is calcium-dependent,hence the advantage of the presence of the ionized calcium incomposition A.

The induction of cell division and of the mitotic index in the germinallayer results in a real restructuring of the epidermis, which becomesmore resistant and deeper, and interacts continually with the dermis,which it protects against dehydration.

2.7.2) At the Dermal Level

The stimulation cascade produced by the local mediators released in theepidermis causes the synthesis of a very abundant extracellular matrixby the fibroblasts. This is very structured. It is in particularcomposed of proteoglycans, of collagen and of adhesion proteins, inparticular of decorin, an adhesion protein which plays a major role inthe phenomena of restructuring of the skin (or skin tissueregeneration). It traps cytokines and other growth factors. Theseobservations were also noted in viva in rats.

Moreover, the enrichment in elastin under the effect of composition A,the fibers of which exhibit better orientation in the connective layer,allow skin tissues to keep their suppleness and their shape during thephysiological degradations caused either by deficiencies and age, or byattacks, in particular stretching.

Turgescent fibroblasts are recruited to the precursor stem cells insufficient number for the effect due to their action and presence to belong-lasting. This effect is maintained by an increasedmicrovascularization.

The skin is more elastic and firmer. It is resistant. Its components arerenewed at a sustained rhythm.

The action of composition A on the various cell types of the skin is oneof repair and regulation of the physiological balance between thevarious constituents. In addition, it has the advantage of being totallyinnocuous and that of having an anti-inflammatory, and thereforesoothing, effect.

EXAMPLE 3 Moisturizing Cream

Moisturizing cream INCI name 100 kg PEG-100 STEARATE GLYCERYL STEARATE1.000 LIQUID PARAFFIN 3.500 LANOLIN 1.700 MYRISTYL ETHOXYMYRISTATE 4.000PLANT OILS 4.000 CETYL ALCOHOL 2.500 TRIETHANOLAMINE 0.200 ANTIOXIDANT0.030 PROPYL PARABEN 0.300 CARBOMERS 0.250 DISODIUM EDTA 0.400 PROPYLENEGLYCOL 2.000 PRESERVING AGENT 0.600 POLYACRYLAMIDE/C13-14 1.000ISOPARAFFIN/LAURETH-7 Composition A 2.500 FRAGRANCE TRACE WATER QS for100

1. A method for preparing a composition, comprising the steps of: a)reducing mother-of-pearl to a powder with to a particle size of betweenapproximately 1 μm and approximately 300 μm; b) bringing mother-of-pearlpowder thus obtained into contact with an extracting agent chosen froman aqueous-glycolic solution of at least one collagen, an aqueousglycolic solution of at least one proteoglycan, or an aqueous glycolicsolution of a mixture of at least one collagen and at least oneproteoglycan, to obtain an extraction mixture; and c) recovering theextraction mixture formed as a result of the bringing into contact. 2.The method as claimed in claim 1, wherein the mother-of-pearl is reducedto a powder with a particle size of between approximately 50 μm andapproximately 100 μm.
 3. The method as claimed in claim 1, wherein themother-of-pearl is reduced to a powder with a particle size of betweenapproximately 15 μm and approximately 50 μm.
 4. The method as claimed inclaim 1, wherein the aqueous-glycolic solution of the extracting agenthas a water:glycol weight ratio of approximately 1:100.
 5. The method asclaimed in claim 1; wherein the collagen is a marine collagen.
 6. Themethod as claimed in claim 1, wherein the collagen concentration isbetween approximately 0.0001% and approximately 50% by weight, relativeto the total weight of the extracting agent.
 7. The method as claimed inclaim 1, wherein the extracting agent is an aqueous-glycolic solution ofat least one proteoglycan.
 8. The method as claimed in claim 7, whereinthe proteoglycan is chosen from the group consisting of hyaluronic acid,chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfateand mixtures thereof.
 9. The method as claimed in claim 7, wherein theextracting agent is an aqueous-glycolic solution of hyaluronic acid. 10.The method as claimed in claim 7, wherein the proteoglycan concentrationis preferably between approximately 0.0001 and approximately 40% byweight, relative to the total weight of the extracting agent.
 11. Themethod as claimed in claim 1, wherein the mother-of-pearl powder isbrought into contact with the extracting agent according to step b) bypreparing a mixture, including the mother-of-pearl powder and theextracting agent, wherein it comprises, relative to its total weight,approximately 20% to approximately 60% by weight of mother-of-pearlpowder obtained in step a) and the remainder as extracting agent. 12.The method as claimed in claim 1, wherein the contact in step b) isbrought about, for a given temperature, for a period of time sufficientto produce a substantially complete extraction.
 13. The method asclaimed in claim 1, wherein at the end of step b), the extractionmixture, formed as a result of the bringing into contact, is recoveredand a liquid phase of the composition is separated from a solid phase.14. A composition obtained using the method as claimed in claim
 1. 15.The composition as claimed in claim 14, in the form of anaqueous-glycolic suspensions, comprising: aragonite (CaCO₃); traceelements chosen from the group consisting of sodium, magnesium,lanthanum, zinc, bromine, cesium, iron, manganese, chlorine, copper,potassium, calcium, strontium, sulfur and mixtures thereof; fibrousproteins from mother-of-pearl; nonfibrous proteins from mother-of-pearl;and at least one collagen not derived from mother of-pearl and/or atleast one proteoglycan not derived from mother-of-pearl.
 16. Thecomposition as claimed in claim 14, comprising at least one marinecollagen not derived from mother-of-pearl and at least one proteoglycannot derived from mother-of-pearl chosen from the group consisting ofhyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate,keratan sulfate and mixtures thereof.
 17. A pharmaceutical composition,comprising the composition as claimed in claim 14, as an activeprinciple, and at least one pharmaceutically acceptable excipient. 18.The pharmaceutical composition as claimed in claim 17, wherein thepharmaceutically acceptable excipient is an excipient suitable fordermatological application.
 19. A therapeutic method-intended for thetreatment of tissue regeneration disorders of skin and/or superficialbody growths, comprising a step of applying to the skin and/orsuperficial body growths a composition as claimed in claim
 14. 20. Atherapeutic method for treatment of disorders of skin and/or superficialbody growths related to aging comprising a step of applying to skinand/or to superficial body growths a composition as claimed in any oneof claims 14 to
 16. 21. A therapeutic method for treatment ofinflammatory skin manifestations comprising a step of applying to skinand/or superficial body growths a composition as claimed in any one ofclaims 14 to
 16. 22. A cosmetic composition, comprising the compositionas claimed in claim 14, as a cosmetically active principle, and acosmetically acceptable excipient.
 23. A cosmeticmethod-intended-for-cosmetic-treatment for tissue regeneration of skinand/or superficial body growths comprising a step of applying to skinand/or to superficial body growths a composition as claimed in claim 14.24. A cosmetic method for cosmetic treatment of modifications related toaging of skin and/or superficial body growths comprising a step ofapplying to skin and/or to superficial body growths a composition asclaimed in claim
 14. 25. A method for preparing a composition,comprising the steps of: a) reducing mother-of-pearl to a powder to aparticle size of between approximately 1 μm and approximately 300 μm; b)bringing mother-of-pearl powder thus obtained into contact with anextracting agent in the form of an aqueous-glycolic solution to obtainan extraction mixture of at least on of a collagen and a proteoglycan;and c) recovering the extraction mixture formed as a result of thebringing into contact.
 26. The method as claimed in claim 1, wherein theaqueous-glycolic solution extracting agent has a water:glycol weightratio of approximately 100:1.